Cholera, a severe acute watery diarrhoeal disease, is caused by a motile, Gram-negative, bacillus named Vibrio cholerae. Millions of people around the globe died of cholera in the past. Cholera has remained endemic in South Asia and the first six pandemics have been considered to have originated from the Bay of Bengal. The favorable climatic conditions and contaminated water and food have maintained the disease in this region including Pakistan. In the last few years, WHO reported a significant increase in cholera cases around the world particularly in Haiti, Zimbabwe and Pakistan. The present study was carried out to characterize Vibrio cholerae isolates from Pakistan which involved determining the prevalence of different serogroups, phenotypic and genotypic characterization of associated antibiotic resistance, analysis of the cholera toxin (CTX) prophage, clonal relationship study, whole genome sequence analysis and single nucleotide polymorphisms (SNPs) based phylogeny. During this study (2009-2011), 113 V. cholerae O1 El Tor isolates were collected from cholera patients in different cities of Pakistan. Among these 113 isolates, 108 (96%) have O1 serogroup and El Tor biotype whereas the serotype was Ogawa. Serogroup O139 which used to exist in Pakistan and elsewhere in the past was replaced by O1 serogroup. All the isolates were resistant to sulfamethoxazole, trimethoprim, streptomycin and nalidixic acid. However, resistance to tetracycline, ampicillin, ceftazidime, erythromycin, cefotaxime, chloramphenicol and ciprofloxacin was seen in 63%, 19.5%, 7.2%, 2%, 01%, 06% and 01% isolates respectively, whereas all isolates were sensitive to ofloxacin. At the genotypic level SXT integrative and conjugative element (ICE), was present in all the isolates whereas integrons (class 1, 2 and 3) and qnrA, qnrB and qnrS for encoding quinolone resistance were absent in all the O1 El Tor isolates studied. Genetic basis of resistance to sulfamethoxazole, trimethoprim, streptomycin and tetracycline was analyzed by detecting sul2, dfrA1, strAB, tetA and tetA which were present in all the isolates showing resistance to the respective antibiotic respectively. florR was detected in about 37 isolates, however only six of them showed resistance phenotype for chloramphenicol. gyrA and parC were also studied for mutations responsible for quinolones resistance; all the isolates had transversions of AGT (underlined) and TCG (underlined) in codons 83 (substituting isoleucine for serine) and 85 (substituting leucine for serine) in case of gyrA and parC respectively, these mutations render bacteria resistant to quinolones. Year wise (2009- 2011) antibiotic analysis showed an increasing trend of antibiotic resistance which should be properly addressed by focusing on the standard treatment of cholera, rehydration therapy, whereas antibiotics should be prescribed only in case of severe dehydration. CTX prophage was analyzed by different PCRs and sequencing approaches. Cholera toxin which is the major virulence factor of V. cholerae was present in all O1 El Tor isolates except one isolate, CS15 from Charsada. All the isolates have ctxB of classical biotype. CTX prophage analysis revealed that all isolates have only one copy of CTXф located on the large chromosome, no tandem repeats of CTX prophage and RS1 were found and the order of RS1 and CTX prophage in the genome of V. cholerae O1 El Tor was: 5’-RSI-CTX prophage-3’. The frequency of heptanucleotide repeat (TTTTGAT) between ctxA and zot for ToxR binding in these isolates varied from 5 to 6 which is high in the region and frequently related to the toxin productivity of the isolates. Multi-locus variable number of tandem repeat analysis (MLVA) of V. cholerae O1 El Tor isolates based on five loci divided the 98 El Tor isolates into 47 sequence types belonging to six clonal complexes (CCs) and three singletons. Epidemiological data revealed that CC1 was associated with cholera cases all over the country in 2011 and Rawalpindi in 2009 whereas as V. cholerae O1 El Tor causing cholera in 2010 were associated with CC2, CC4 and CC3. Based on characteristic antibiotic resistance patterns and presence/absence of tagA and aldA, all V. cholerae O1 El Tor isolates were categorized in two groups, however MLVA generated clonal complexes did not reflected such relationship. The whole genome sequence analysis of the isolates and comparative genomics divided the V. cholerae O1 El Tor isolates from Pakistan in two categories. Genome wide SNPs analysis was carried out using the whole genome sequence data and a global phylogenetic tree was constructed comparing Pakistan Vibrio cholerae O1 El Tor isolates with 146 global and temporal representative V. cholerae isolates. All the O1 El Tor isolates from Pakistan were classified in two unique sub-clades named as Pakistan sub-clade 1 (PSC-1) and Pakistan sub-clade 2 (PSC-2) respectively. Both PSCs belonged to the third transmission wave of the current seventh pandemic. Both sub-clades possessed distinct antibiotic resistance patterns and were distinguished by signature deletions in Vibrio pathogenicity island -1 (VPI-1) and Vibrio seventh pandemic 2 (VSP- 2). All the PSC-1 isolates had a unique three gene (VC0819-VC0821) deletion in VPI-1 whereas in PSC-2 VPI-1 was intact. In PSC-1 a four gene (VC0495-VC0498) deletion was present in VSP-2 whereas a large 18 gene (VC0495-VC0512) deletion was present in VSP-2 of PSC-2. PSC-2 representing (4/4) and (31/38) isolates in 2009 and 2010 respectively was dominant in Pakistan whereas PSC-1 was only seen in Karachi representing (6/7) isolates. However in 2011, PSC-1 has apparently replaced PSC-2 representing 54/56 (96.5%) isolates and only 2/56 (3.5%) belonged to PSC-2. In nutshell, the study showed that two sub-clades with distinct antibiotic resistance patterns and genomic signatures circulating in Pakistan caused cholera during 2009-2011. Furthermore, SNPs based genetic markers can be used to track and identify the distribution of existing V. cholerae sub-clades or even any new type in future.